Abstract:

Enhancement of fluorescence emission from fluorophores bound to a sample
and present on the surface of two-dimensional photonic crystals is
described. The enhancement of fluorescence is achieved by the combination
of high intensity near-fields and strong coherent scattering effects,
attributed to leaky photonic crystal eigenmodes (resonance modes). The
photonic crystal simultaneously exhibits resonance modes which overlap
both the absorption and emission wavelengths of the fluorophore. A
significant enhancement in fluorescence intensity from the fluorophores
on the photonic crystal surface is demonstrated.

Claims:

1. A detection instrument for a biosensor, comprising:an optical system
directing light onto the biosensor comprising a laser light unit, a beam
expander expanding a beam generated by the laser light unit, and a device
for adjusting polarization of the light generated by the laser light
unit;an angle-tunable mirror receiving beam-expanded laser light and
directing the expanded beam to the biosensor; andan imaging system
receiving light from biosensor, the imaging system comprising an optical
path containing an objective lens, a narrow-linewidth bandpass filter and
a camera.

2. The detection instrument of claim 1, wherein the angle-tunable mirror
is mounted to a motorized stage.

3. The detection instrument of claim 1, wherein the objective lens
comprises the objective lens of a microscope and wherein the biosensor is
in the form of a photonic crystal formed in a microscope slide.

4. The detection instrument of claim 1, wherein the wavelength of the
laser light unit is tuned to match the excitation band of a fluorophore
present in a sample placed on the biosensor.

5. The detection instrument of claim 4, wherein the frequency of the
bandpass filter coincides with a resonance frequency exhibited by the
biosensor.

7. The detection instrument of claim 1, further comprising a variable
neutral density filter in the optical system directing light onto the
biosensor attenuating the output of the laser light unit.

8. The detection instrument of claim 1, further comprising a brightfield
illumination source for illuminating the biosensor, and a second camera
capturing magnified brightfield images of the biosensor.

9. The detection instrument of claim 1, further comprising a low numerical
aperture lens in the optical system directing light onto the biosensor,
wherein the tunable mirror receives light from the low numerical aperture
lens.

10. The detection instrument of claim 1 further comprising a dichoric
mirror reflecting laser light passing through the biosensor and a
photodiode detector receiving the reflected laser light.

11. The instrument of claim 1, wherein the biosensor in is the form of a
microwell plate having a plurality of wells, the wells having a photonic
crystal sensor formed therein.

12. The instrument of claim 1, wherein the angle-tunable mirror is moved
such that the camera captures an image of the biosensor at an angle of
minimum transmission.

13. The instrument of claim 1, wherein the biosensor comprises a photonic
crystal and, wherein a sample containing a fluorophore is applied to the
biosensor, and wherein the light from the biosensor comprises enhanced
fluorescence from the fluorophore, the fluorophore be excited by the
light from the laser light unit.

14. A detection system for a biosensor comprising a photonic crystal
incorporated into a test device, combining in combination a microscope
and the detection instrument of claim 1, wherein the objective lens
comprises the objective lens of the microscope.

15. A detection instrument for a photonic crystal biosensor exhibiting an
enhanced excitation and extraction modes, comprising:a laser light
source;an optical system directing light from the laser light source to
the photonic crystal biosensor, the biosensor responsively and
simultaneously exhibiting (1) an excitation resonance mode having a
spectrum which at least partially overlaps an excitation spectrum of a
fluorophore bound to the biosensor; and (2) an extraction resonance mode
having a spectrum which at least partially overlaps the emission spectrum
of the fluorophore, anda detection system placed to capture light from
the biosensor in the extraction resonance mode.

16. The instrument of claim 15, wherein the detection system includes a
camera generating an image of the biosensor.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This is a continuation of U.S. application Ser. No. 11/986,156 filed
Nov. 19, 2007, which claims priority under 35 U.S.C. §119(e) to U.S.
Provisional Application Ser. No. 60/916,462 filed May 7, 2007, the
content of which is incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0003]Photonic crystals, also commonly referred to as photonic bandgap
structures, are periodic dielectric structures exhibiting a spatially
periodic variation in refractive index that forbids propagation of
certain frequencies of incident electromagnetic radiation. The photonic
band gap of a photonic crystal refers to the range of frequencies of
electromagnetic radiation for which propagation through the structure is
prevented in particular directions. A photonic crystal structure may be
designed to exhibit extraordinarily high reflection efficiency at
particular wavelengths, at which optical standing waves develop and
resonate within the photonic crystal structure. Such optical resonances
are known to occur at the wavelengths adjacent to the photonic band gap,
sometimes referred to as the photonic band edge. The spatial arrangement
and refractive indices of these structural domains generate photonic
bands gaps that inhibit propagation of electromagnetic radiation centered
about a particular frequency.

[0004]This anomalous resonant phenomenon (termed guided-mode resonance)
arises due to the introduced periodicity which allows phase-matching of
externally incident radiation into modes that can be reradiated into
free-space. Due to the fact that these modes possess finite lifetimes
within such structures, they are referred to as `leaky eigenmodes` of the
structures. More recently, guided-mode resonances have been studied in
crossed gratings or two-dimensional (2D) photonic crystal (PC) slabs. The
leaky nature of these modes has been exploited towards the development of
light emitting diodes (LEDs) with improved extraction efficiency,
biosensors (see Cunningham et al., Colorimetric Resonant Reflection as a
Direct Biochemical Assay Technique, Sensors and Actuators B, 2002, 81,
pgs 316-328 (2002)) and vertically emitting lasers.

[0005]The ability of photonic crystals to provide high quality factor (Q)
resonant light coupling, high electromagnetic energy density, and tight
optical confinement can also be exploited to produce highly sensitive
biochemical sensors. Here, Q is a measure of the sharpness of the peak
wavelength at the resonant frequency. Photonic crystal biosensors are
designed to allow a liquid test sample to penetrate the periodic lattice,
and to tune the resonant optical coupling condition through modification
of the surface dielectric constant of the crystal through the attachment
of biomolecules or cells. Due to the high Q of the resonance, and the
strong interaction of coupled electromagnetic fields with surface-bound
materials, several of the highest sensitivity biosensor devices reported
are derived from photonic crystals. Such devices have demonstrated the
capability for detecting molecules with molecular weights less than 200
Daltons (Da) with high signal to noise margins, and for detecting
individual cells. Because resonantly coupled light within a photonic
crystal can be effectively spatially confined, a photonic crystal surface
is capable of supporting large numbers of simultaneous biochemical assays
in an array format, where neighboring regions within ˜10 μm of
each other can be measured independently. See Li, P., B. Lin, J.
Gerstenmaier, and B. T. Cunningham, A new method for label-free imaging
of biomolecular interactions. Sensors and Actuators B, 2003.

[0006]Given substantial advances in their fabrication and their unique
optical properties, photonic crystal-based sensors are under development
for a variety of applications. Biosensors are one application. Biosensors
incorporating photonic crystal structures are described in the following
references, which are hereby incorporated by reference in their
entireties: U.S. Pat. Nos. 7,118,710, 7,094,595, and 6,990,259; U.S.
Published applications 2007/0009968; 2002/0127565; 2003/0059855;
2007/0009380; 2003/0027327; Cunningham, B. T. J. Qiu, P. Li, J. Pepper
and B. Hugh, A Plastic calorimetric Resonant Optical Biosensor for
Multi-parallel Detection of Label Free Biochemical Interactions, Sensors
and Actuators B, 2002, 85, pgs 219-226.

[0007]U.S. Pat. No. 6,707,561 describes a grating-based biosensing
technology that is sometimes referred to in the art as Evanescent
Resonance (ER) technology. This technology employs a submicron scale
grating structure to amplify a fluorescence signal, following a binding
event on the grating surface, where one of the bound molecules carries a
fluorescent label. ER technology enhances the sensitivity of fluorophore
based assays enabling binding detection at analyte concentrations
significantly lower than non-amplified assays.

[0008]ER technology uses grating generated optical resonance to
concentrate laser light on the grating surface where binding has taken
place. In practice, a laser scanner sweeps the sensor at some angle of
incidence (θ), typically from above the grating, while a detector
detects fluoresced light (generally at longer optical wavelength) from
the sensor surface. By design, ER grating optical properties result in
nearly 100% reflection, also known as resonance, at a specific angle of
incidence and laser wavelength (λ). Confinement of the laser light
by and within the grating structure amplifies emission from fluorophores
bound within range of the evanescent field (typically 1-2 μm). Hence,
at resonance, transmitted light intensity drops to near zero.

[0009]The spectral width and wavelength of the resonance phenomena
describes the important externally measurable parameters of a device.
Resonance width refers to the full width at half maximum, in wavelength
measure, of a resonance feature plotted as reflectance (or transmittance)
versus wavelength. Resonance width also refers to the width, in degrees,
of a resonance feature plotted on a curve representing reflectance or
transmittance as a function of θ. In practice, one can make
adjustments to the incident angle to "tune" the resonance towards maximum
laser fluorophore coupling.

[0010]In one embodiment of this invention, a biosensor is constructed as a
photonic crystal structure which has a periodic surface grating in which
a so-called evanescent resonance is created. Conceptually, resonance
phenomena can occur in planar dielectric layer gratings where almost 100%
switching of optical energy between reflected and transmitted waves
occurs when the grooves of the grating have sufficient depth and the
radiation incident on the corrugated structure is at a particular angle.
This phenomenon is exploited in the sensing area of the platform where
that sensing area includes grating structures (e.g., grooves, or holes or
posts) of sufficient depth and light is caused to be incident on the
sensing area of the platform at an angle such that evanescent resonance
occurs in that sensing region. This creates in the sensing region an
enhanced evanescent field which is used to excite samples under
investigation. It should be noted that the 100% switching referred to
above occurs with parallel beam and linearly polarized coherent light and
the effect of an enhanced evanescent field can also be achieved with
non-polarized light of a non-parallel focused laser beam. Excitation
photons incident on the sample (chip, for example) under resonance
conditions couple into a thin corrugated surface (such as a metal oxide
layer) at the site of incidence. As a result of the transducer geometry,
the energy is locally confined into the thin corrugated layer of high
refractive index material. Consequently, strong electromagnetic fields
are generated at the surface of the chip. The effect has been attributed
as evanescent resonance and leads to increased fluorescence intensity of
chromophores (fluorescent material) close to the surface of the sensor.
The effective field strength can be increased up to 100-fold by the
confinement of the available excitation energy, depending on the optical
properties of the optical detection system used.

[0011]The inventive sensors and method of this disclosure are useful in
conjunction with a variety of different types of fluorophores. Such
fluorophores have excitation and emission spectra which are typically
well characterized and available from the manufacturer, or can be
determined experimentally.

[0012]Quantum Dots (QDs) are fluorescent, nanometer-sized inorganic
semiconductor crystals that have rapidly emerged as an important class of
nanomaterials which promise to revolutionize a wide range of
nanotechnology-enabled fields. QDs derive their unique optical properties
(broad absorption spectrum, narrow, size-tunable emission spectrum, high
photostability, quantum efficiency and strong nonlinear response) from
quantum confinement effects. These attributes, coupled with the ability
to functionalize QDs, has made them important candidates for light
sources, solar cells, optical switches and fluorescent probes in
sensitive biological assays. The ability to more efficiently excite and
extract the light emitted by QDs would thus be of vital importance in
realizing high brightness light sources, enhanced nonlinear effects and
lowering the detection limits in biological assays.

[0013]Fluorescent dyes represent a broad class of organic and inorganic
fluorescent molecules that are capable of emitting light. Generally,
electrons within the fluorescent molecule are excited from a ground state
to an excited state through the absorption of a photon from an external
source of illumination. The electron in the excited state may return to
the ground state through a variety of mechanisms. One such mechanism is
through the release of heat in the form of a phonon. Another such
mechanism is through the release of light in the form of a photon.
Absorption of energy by the fluorophore occurs at a particular range of
incident photon energies (or equivalently wavelengths) that are unique
for each type of molecule. Due to conservation of energy, the emitted
photon energy must be less than or equal to the energy of the incident
photon, and therefore the emitted wavelength must be larger than the
incident photon wavelength. Therefore, a fluorescent molecule has two
distinct spectra associated with it: the range of wavelengths for which
it is capable of absorbing photons, and the range of wavelengths for
which it is capable of emitting photons. The difference between the
absorption and emission wavelength is known as the Stokes shift.

SUMMARY

[0014]Photonic crystal sensors are disclosed for use in testing samples in
which a fluorophore, e.g., inorganic crystalline semiconductor ("quantum
dot") or fluorescent dye is present in the sample. The sample and
fluorescent dye are in close proximity, or more typically bound, to the
photonic crystal surface, e.g., by depositing the sample with fluorophore
on the sensor surface in a dry or aqueous environment.

[0015]In one aspect of this disclosure, the photonic crystal sensor is
constructed and arranged with a surface in the form of a periodic surface
grating structure which simultaneously exhibits multiple resonance modes
for light at a given incident angle. The resonance modes overlap both the
excitation and emission spectra of the fluorophore. In particular, when
light is incident upon the photonic crystal at an appropriate incident
angle θ, the photonic crystal sensor simultaneously exhibits
multiple resonance modes (referred to herein as leaking eigenmodes or
leaky modes) which have spectra that overlap both the absorption
(excitation) and emission spectra of the fluorophore present in the
sample. In this document, the term "spectrum" in the context of a
resonant mode refers to the band of wavelengths of incident light in
which a guided mode resonance is created in the photonic crystal as the
angle of incidence θ varies. A photonic crystal constructed and
arranged so as to possess such a doubly resonant scheme (i.e., exhibiting
resonance modes overlapping both the excitation and emission spectra of
the fluorophore simultaneously at a given incident angle θ) yields
strongly enhanced fluorescent emission and the ability to extract such
emission in a highly efficient manner, resulting in a high sensitive
sensors suitable for a very broad range of applications, as will be
explained below.

[0016]A sample testing system for testing a sample having a fluorophore
bound to the sample is also described. The testing system includes a
detection instrument comprising a light source and a detector; and a
photonic crystal sensor having a periodic grating structure. The sample
including the fluorophore is placed on the periodic grating structure.
The light source of the detection system is oriented relative to the
photonic crystal sensor such that the light source illuminates the
photonic crystal sensor at a incident angle θ in which the photonic
crystal simultaneously exhibits a plurality of resonant modes, the
resonant modes including an excitation mode having a first resonant
spectrum and an extraction mode having a second resonant spectrum. The
periodic grating structure is constructed and arranged such that the
resonant spectrum of the photonic crystal in the excitation mode at least
partially overlaps the excitation spectrum of the fluorophore and the
resonant spectrum in the extraction mode at least partially overlaps the
emission spectrum of the fluorophore. The detector operates to detect
radiation from the fluorophore in the emission spectrum.

[0017]A method of testing a sample with a fluorophore present in the
sample with the photonic crystal sensors of this disclosure are also
described. The method includes the step of placing the sample onto the
surface of a photonic crystal sensor; illuminating the photonic crystal
biosensor with light at an angle of incidence θ, the biosensor
responsively and simultaneously exhibiting (1) an excitation resonance
mode having a spectrum which at least partially overlaps the excitation
spectrum of the fluorophore; and (2) an extraction resonance mode having
a spectrum which at least partially overlaps the emission spectrum of the
fluorophore, the illumination and the resulting excitation and extraction
resonance modes causing the fluorophore to emit light; and collecting the
emitted light from the fluorophore and directing the emitted light onto a
detector.

[0018]In yet another aspect, a photonic crystal sensor is disclosed which
includes a periodic surface grating structure which exhibits a resonance
mode at a given incident angle which overlaps the emission spectrum of a
fluorophore which is present with a sample deposited on the sensor, and
does not have a resonance mode which overlaps the excitation spectrum of
the fluorophore. The photonic crystal sensor produces an enhanced
extraction effect without also producing an enhanced excitation effect.
Sample testing systems suitable for the doubly resonant photonic crystal
sensors are also useful with this embodiment.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1 is an illustration of a photonic crystal featuring the
enhanced excitation and extraction features of this disclosure and
comparing the directionally enhanced extraction of emitted radiation from
fluorophores from the photonic crystal with a dielectric slab which does
not feature the resonance modes of this disclosure.

[0020]FIG. 2a is an illustration of a two-dimensional photonic crystal
device having a periodic surface grating structure constructed as a two
dimensional array of holes in accordance with one exemplary embodiment of
the invention. FIG. 2B shows Scanning Electron Micrograph (SEM) images of
a cleaved photonic crystal device of FIG. 2a. In FIG. 2a, the lines
joining the features Γ, X and M are axes of high symmetry in the
photonic crystal surface. Λ=300 nm is the period of the surface
grating structure (with a square unit cell in X and Y directions) and
t=125 nm, the thickness of a high index of refraction material layer
which is deposited onto the grating layer. Θ is the angle the
incident beam of light makes with the vertical.

[0021]FIGS. 3a and 3b are graphs of the calculated and experimental
dispersion spectra, respectively, of the fabricated photonic-crystal
sensor of FIGS. 2a and 2b for white, S-polarized incident illumination
incident along the δ-M direction. The sensor exhibits resonant mode
at λ=488 nm when the incident beam makes an angle
θ=11.2° with the surface normal. The excitation of the
resonant mode at this wavelength, where the quantum dot fluorophore is
strongly absorbing, provides the required near-field enhancement for
evanescent resonance (enhanced excitation). The shading scale shows the
efficiency of transmission. Higher Q factors for a resonance mode are
indicated by thinner lines whereas broader Q factors for a resonance mode
are indicated by relatively thicker lines.

[0022]FIGS. 4a and 4b are calculated near electric-field intensities
(E2) at resonance for the photonic crystal structure of FIG. 2a,
with FIG. 4a showing the intensity for the lower surface of the surface
grating (bottom of the hole) and FIG. 4b showing the intensity for the
upper surface of the surface grating. The intensities are for the leaky
mode (resonance) with λ=488 nm when the incident beam makes an
angle θ=11.2° with the surface normal. The intensity is
normalized to the unit amplitude incident wave.

[0023]FIGS. 5a, 5b and 5c are graphs of the dispersion spectrum showing
the resonance modes of the photonic crystal sensor showing the
possibility of enhanced extraction for all polarizations and directions
of incident white light. The graphs were experimentally determined from
the photonic crystal of FIG. 2a. FIG. 5a is the graph for P-polarized and
incident along the Γ-M direction, FIG. 5b is the graph for
P-polarized and incident along the Γ-X direction, and FIG. 5c is
the graph for S-polarized, incident along Γ-X direction.

[0024]FIGS. 6a and 6b are fluorescence (pseudocolor) scan images of the
photonic crystal of FIG. 2a with quantum dots dispensed on the surface.
FIG. 6a is a scan image taken when the photonic crystal is resonant with
respect to the incident beam (θ=11.2°), showing an
enhancement factor of over 108 times. FIG. 6b is a scan image taken when
the photonic crystal is not resonant with the incident beam
(θ=0°), showing an enhancement factor of over 13 times. The
circular regions represent the area over which intensity information was
averaged. In both the images, the circle to the left shows the control
region where no photonic crystal is present.

[0025]FIG. 7 is a near-field scanning optical microscopy (NSOM) image of
the near-fields on the surface of a fabricated photonic crystal device.

[0026]FIG. 8 is an illustration of an angle-resolved fluorescence
measurement from the photonic crystal surface for the Γ-X
direction.

[0027]FIG. 9 is a graph of the normalized fluorescence spectrum of a
quantum dot fluorescence as modified by the photonic crystal sensor.

[0028]FIG. 10 illustrates angle-resolved fluorescence measurements from
the photonic crystal surface for the light polarized in the Γ-M
direction.

DETAILED DESCRIPTION

[0029]Photonic crystal sensors are disclosed for use in testing samples in
which a fluorophore, e.g., inorganic crystalline semiconductor ("quantum
dot") or fluorescent dye is present in the sample. The sample and
fluorescent dye are in close proximity, or more typically bound, to the
to the photonic crystal surface, e.g., by depositing the sample with
fluorophore on the sensor surface in a dry or aqueous environment. An
example of the photonic crystal is shown in FIG. 2a and will be described
in detail subsequently.

[0030]In one aspect of this disclosure, the photonic crystal sensor is
constructed and arranged with a surface in the form of a periodic surface
grating structure (such as a two dimensional array of holes shown in FIG.
2a), which simultaneously exhibits multiple resonance modes for light at
a given incident angle. The resonance modes are shown graphically in
FIGS. 4 and 6 (transmission efficiency shown plotted as a function of
wavelength for incident light at different angles to vertical), described
subsequently, and are indicated in the areas of the graphs where the
transmission efficiency drops to zero. The resonance modes overlap both
the excitation and emission spectra of the fluorophore present in the
sample. In particular, when light is incident upon the photonic crystal
at an appropriate incident angle θ, the photonic crystal sensor
simultaneously exhibits multiple resonance modes (referred to herein as
leaking eigenmodes or leaky modes) which have spectra that overlap both
the absorption (excitation) and emission spectra of the fluorophore
present in the sample. A photonic crystal constructed and arranged so as
to possess such a doubly resonant scheme (i.e., exhibiting resonance
modes overlapping both the excitation and emission spectra of the
fluorophore) to enhance fluorescent emission from the fluorophore has
not, to the current knowledge of the inventors, been previously
demonstrated.

[0031]The presence of the photonic crystal resonance peak occurring at the
fluorescence excitation wavelength gives rise to the formation of high
intensity electromagnetic near-fields which serves to efficiently excite
the fluorophore present in the sample. This phenomenon is referred to
herein interchangeably as "evanescent resonance" or "enhanced
fluorescence."

[0032]The additional feature of a resonance mode in the photonic crystal
which overlaps the emission spectrum of the fluorophore serves as an
effective mechanism to extract this enhanced emission. In particular, a
photonic crystal with resonance modes overlapping both the emission and
excitation spectra of the fluorophore increases the number of
fluorescence emitted photons which can be gathered by a detector in an
associated measuring instrument for use with the photonic crystal sensor.
Photonic crystal resonance occurring at the emission wavelength of the
fluorophore can be used to efficiently couple emitted photons at the
photonic crystal surface to be selectively directed into free space at a
particular exit angle, instead of uniformly directed in all directions.
This phenomenon of selective direction of emitted photons is believed to
be due to fluorescence coupling to the overlapping leaky modes producing
Bragg scattering out of the structure, thereby greatly reducing the
amount of light trapped in the photonic crystal sensor in the extraction
mode. If the dispersion of these overlapping emission leaky modes is
close to the Γ-point band edge (i.e., the magnitude of the in-plane
wave vector for incident polarized light is close to zero), a significant
amount of the emitted light can be extracted from the photonic crystal
sensor within small angles of the vertical. This discovery allows for
positioning of a detector (or associated optical elements such as fiber
optic probe which is coupled to a detector) at the correct position
relative to the photonic crystal surface, and allows for capturing more
photons that would otherwise occur, e.g., as compared to a fluorophore
emitting from a non-photonic crystal surface.

[0033]A photonic crystal sensor as just described is shown in FIG. 1 as
item 10, with a dielectric slab 20 shown next to it which does not
possess a photonic crystal property for purposes of comparison. The
photonic crystal sensor 10 consists of a periodic surface grating
structure formed on its surface 11, which in this example takes the form
of a two-dimensional array of holes 12. (Other periodic structures for
the photonic crystal 10 are possible, as will be explained below). A
sample containing a fluorophore 14 is applied to the surface 11. The
photonic crystal is illuminated with light (in this case from above) at
an angle θ relative to the vertical direction. The angle θ is
shown in FIG. 2a. The properties of the periodic grating (holes) 14 on
the surface 11 are such that the photonic crystal simultaneously exhibits
resonance modes which overlap both the excitation and emission spectra of
the fluorophore 14. The overlap of the resonance mode with the excitation
spectrum produces an enhanced excitation of the fluorophore 14. The
overlap of the resonance mode with the emission spectrum of the
fluorophore 14 produces a directionally enhanced extraction of the
emitted radiation due to Bragg scattering, which is indicated by the
lines 16 all pointing in the same direction. As a practical matter, the
angle of the lines 16 can be determined either by simulation or by
experimentation and light collection apparatus placed in alignment with
the lines 16 so as to collect this radiation. The light collection
apparatus (e.g., fiber optic probe) supplies the collected radiation to a
detection device such as a CCD imager or photomultiplier tube so as to
make measurements or collect images of the radiation, thereby obtaining
information as to the sample.

[0034]Consider now the dielectric slab 20 in FIG. 1 which does not possess
any photonic crystal attributes for the sake of comparison. Assuming the
incident light encompasses the excitation spectrum of the fluorophore 14,
at least some fluorescence can be expected. However, because there is no
resonant mode in the slab 20 overlapping the emission spectrum of the
fluorophore, the resulting fluorescence is radiated in an almost
spatially uniform manner, indicated by the lines 16 pointing in all
directions. Thus, the concentration of radiation in the same direction as
indicated at 16 on the left hand side of FIG. 1 produces an enhanced
extraction of the fluorescent signal which is not present in a
non-photonic crystal structure such as shown on the right hand side of
FIG. 1. (While light can be directed onto the surface 11 from above, as
shown in FIG. 1, it can also be incident on the photonic crystal from
below and the same resonant effects are produced in the photonic
crystal).

[0035]The gain in sensitivity in the photonic crystal sensor 10 of FIG. 1
obtained by (1) evanescent resonance (enhanced fluorescence due to one
resonance mode of the photonic crystal overlapping the excitation or
absorption spectrum of the fluorophore) and (2) enhanced extraction (due
to another resonance mode of the photonic crystal overlapping the
emission spectrum of the fluorophore) are multiplied together to derive
the overall gain in sensitivity resulting from the combination of these
features. For example, if the evanescent resonance provides for a
hundred-fold increase in the amount of fluorescence emission, and the
enhanced extraction provides the ability of the detector to collect a
five times as many fluorescent photons, then the overall sensitivity of
an assay performed on a photonic crystal surface using the combined
techniques will be five hundred (500) times greater than the same assay
performed on an ordinary surface (e.g., microscope slide, microplate or
microfluidic flow channel) which does not possess the photonic crystal
properties of this invention.

[0036]Thus, one benefit of a photonic crystal sensor 10 with the doubly
resonant properties as described above is that it provides a sensor
platform in which a very substantial increase in the sensitivity of
fluorescent assays occurs than would otherwise be possible. As will be
appreciated from the following detailed description, photonic crystal
sensors in accordance with the invention are useful in a number of
different applications. These include:

[0037]1) Gene expression microarrays incorporating the photonic crystal
sensor 10. The genes may be detected at lower expression levels and or
with smaller sample volumes than previously known.

[0038]2) Protein detection assays, such as for example detection of
protein biomarkers in bodily fluids for disease diagnostic tests, where
proteins are present in very low concentrations. Detection by the methods
of this disclosure would be more sensitive than commonly use ELISA
assays, but with a simpler assay protocol.

[0039]3) Fluorescent imaging of cells, viruses, tissue samples, bacteria,
proteins etc., using a microscope. The photonic crystal sensor 10 of this
disclosure can be incorporated onto the surface of a microscope slide. A
specimen is stained with one or more fluorescent dyes or quantum dot
fluorophores (which may be conjugated to an antibody or other probe of
interest) and placed on the slide such that the specimen and
fluorophore(s) are in contact with the surface of the photonic crystal
sensor 10. The improved sensitivity can be used to observe dye molecules
at lower concentrations and/or to use lower cost imaging cameras with an
improved signal to nose ratio.

[0040]4) The photonic crystal sensor 10 can be produced uniformly over
large surface areas using a nano-replica molding process which is
suitable for mass production at low cost. The photonic crystal structure
thus produced can be incorporated into the surface of various assay or
testing devices of conventional formats, such as, for example, (1)
incorporation onto the surface of a microscope slide, (2) incorporation
within a standard format microplate, e.g., at the bottom thereof, or (3)
incorporation into any other fluorescent assay format either now known or
later developed.

[0041]Thus, in one embodiment of the invention, a sensor 10 is descried
herein which is adapted to test a sample having a fluorophore present in
the sample which is deposited on the sensor surface. The fluorophore
(e.g. quantum dot, fluorescent dye such as Cy5) has an excitation
spectrum and an emission spectrum. The sample may be placed on the sensor
10 in a dry or an aqueous environment. The sensor includes a photonic
crystal having a periodic grating structure (12). The photonic crystal
exhibits a plurality of resonant modes when illuminated with light at an
incident angle θ. The resonant modes include an excitation mode
having a first resonant spectrum and an extraction mode having a second
resonant spectrum. The periodic grating structure is constructed and
arranged such that the first resonant spectrum of the photonic crystal in
the excitation mode at least partially overlaps the excitation spectrum
of the fluorophore and the second resonant spectrum of the photonic
crystal in the extraction mode at least partially overlaps the emission
spectrum of the fluorophore.

[0042]In one embodiment, the extraction resonant mode of the photonic
crystal at the incident angle exhibits a relatively low Q factor, i.e.,
one in which the Q factor is less than 100. The Q factor of the
extraction mode will determine the rate at which coupled radiation will
be scattered into free space, such as in the case where the fluorophore
exhibits a broad emission spectrum. In other embodiments, the extraction
resonant mode has a Q factor is relatively high, i.e., between 100 and
1000. With a relatively high Q factor, a detector can obtain enhanced
extraction from a narrow band of wavelengths, but with amplified
extraction efficiency. A sensor constructed to produce the optimum Q
factor at the extraction resonant mode for a given application will
depend on several factors, such as

[0043]1) whether the sensor is designed to be used in a detection
instrument which performs single-point detection (with a photomultiplier
tube, as an example) where one can gather a broad range of wavelengths
with a broad range of exit angles, situations where a low Q factor for
the extraction resonant mode might be best, and

[0044]2) whether the sensor is designed to be used in a detection
instrument which performs imaging detection (e.g., with a CCD camera), in
which case a relative high Q factor would provide detection of a narrow
range of wavelengths and a narrow range of exit angles for that
wavelength. The Q factor for the extraction resonance mode can be changed
by changing the parameters of the surface grating structure and simulated
in the design phase as will be described below.

[0045]The sensor as described herein can be incorporated into testing
platforms suited to a variety of specific applications. In one example,
the sensor is incorporated into a gene expression microarray device. In
another example, the sensor is incorporated into a protein detection
assay device and the sample is in the form of a protein. In still other
examples, the sensor is incorporated into a testing device, such as a
microscope slide, which is used to perform fluorescent image analysis of
cells, viruses, bacteria, spores, and tissue samples. In still another
example the sensor is incorporated into a microwell plate having a
plurality of individual sample wells. Each of the sample wells includes a
photonic crystal as described herein.

[0046]In one particular embodiment, the periodic surface grating structure
of the photonic crystal is constructed as a grating layer arranged as a
two-dimensional array of holes each having a depth D, and a relatively
high index of refraction material of thickness t deposited on the grating
layer. Suitable high index of refraction materials include titanium oxide
(TiO2), silicon nitride, hafnium oxide, zinc sulfide, tantalum oxide
and zinc selenide. In preferred embodiments the array of holes has an
axis of symmetry which is either perpendicular or parallel to the
polarization state of the incident light. In FIG. 2a, the lines
connecting the features Γ, X and M are axis of high symmetry in the
photonic crystal surface. The grating layer is positioned above a
substrate layer. The substrate layer may consist of a layer of glass,
quartz, polymers, plastic, polyethylene terepthalate (PET) and
combinations thereof.

[0047]The depth D of the holes can be selected such that the photonic
crystal exhibits the excitation and extraction resonance modes having
spectra which are spectrally separated from each other and which
substantially overlap the excitation and emission spectra, respectively,
of a predetermined fluorophore, such as a particular quantum dot or group
of quantum dots or other fluorophores having similar excitation and
emission spectra.

[0048]In another aspect of this invention, a sample testing system is
described for testing a sample having a fluorophore present in the sample
which is deposited on the photonic crystal sensor. The fluorophore has an
excitation spectrum and emits fluorescence in an emission spectrum. The
sample testing system includes a detection instrument comprising a light
source and a detector, and a photonic crystal sensor comprising a
periodic grating structure. The sample including the fluorophore are
placed on the periodic grating structure.

[0049]The particular construction of the detection system is not
particularly important and can vary widely, depending on the particular
application. Examples of suitable detection systems include those systems
described in U.S. Pat. Nos. 7,118,710, 7,094,595, and 6,990,259 and U.S.
Published applications 2007/0009968; 2002/0127565; 2003/0059855;
2007/0009380; and 2003/0027327.

[0050]The light source of the detection system is oriented relative to the
photonic crystal sensor such that the light source illuminates the
photonic crystal sensor at a incident angle θ in which the photonic
crystal simultaneously exhibits a plurality of resonant modes, the
resonant modes including an excitation mode having a first resonant
spectrum and an extraction mode having a second resonant spectrum.

[0051]The periodic grating structure is constructed and arranged such that
the first resonant spectrum of the photonic crystal in the excitation
mode at least partially overlaps the excitation spectrum of the
fluorophore and wherein the second resonant spectrum of the photonic
crystal in the extraction mode at least partially overlaps the emission
spectrum of the fluorophore.

[0052]In one embodiment, the detector may take the form of an imaging
detector, e.g., charge coupled device (CCD) camera. Other types of
detectors are also possible, including photomultipliers. In preferred
embodiments the light source may be a laser or a broad spectrum source.
The light from the source may be polarized. In one embodiment, the
grating structure of the photonic crystal has an axis of symmetry which
is substantially parallel or perpendicular to the polarization state of
the incident light.

[0053]The sensor can be incorporated into a variety of different testing
device formats, as explained herein, such as a gene expression microarray
device, a protein detection assay device, a microscope slide, and a
microwell plate or dish having a plurality of individual sample wells, in
which each of the sample wells includes a photonic crystal described
herein.

[0054]In a further aspect, a method is disclosed of testing a sample
having a fluorophore bound to the sample, the fluorophore having an
excitation spectrum and an emission spectrum, comprising the steps of:
(a) placing the sample onto the surface of a photonic crystal sensor, (b)
illuminating the photonic crystal biosensor with light at an angle of
incidence θ, the biosensor responsively and simultaneously
exhibiting (1) an excitation resonance mode having a spectrum which at
least partially overlaps the excitation spectrum of the fluorophore; and
(2) an extraction resonance mode having a spectrum which at least
partially overlaps the emission wavelength distribution of the
fluorophore, the illumination and the resulting excitation and extraction
resonance modes causing the fluorophore to emit light, and (c) collecting
the emitted light from the fluorophore and directing the emitted light
onto a detector.

Example

[0055]FIG. 2a is an illustration of a photonic crystal sensor 10 in
accordance with one embodiment of the invention. The sensor 10 includes a
glass substrate layer 22, a grating layer 24 providing a periodic grating
structure (in this case a two dimensional array of holes 12) and a high
index of refraction later 26 of TiO2 which is deposited on the
grating layer 24.

[0056]In order to design a PC that can support multiple guided-mode
resonances, a two-dimensional structure with a sufficiently large
effective index and the features arranged as a square lattice of holes
was chosen, as shown in FIG. 2a. The period (Λ) of the structure
was chosen such that it supports a relatively high Q-factor resonant
modes at a wavelength where the fluorophores (in this instance, quantum
dots) are excited (λ=488 nm, excitation mode) and low Q-factor
modes overlapping the quantum dot fluorescence emission spectrum
(centered at λ=616 nm, extraction mode). In FIG. 2a, the features
Γ, X and M are points of high symmetry in the photonic crystal
surface. Λ=300 nm is the period of the surface grating structure
(with a square unit cell in X and Y directions) and t=125 nm, the
thickness of a high index of refraction material layer 26 which is
deposited onto the grating layer 24. Θ is the angle the incident
beam of light 13 makes with the vertical (direction normal to the surface
11).

[0057]The logic governing the choice of low Q-factor extraction modes for
some embodiments will become clear shortly, and although a relatively low
Q-factor resonance mode at the emission spectrum is shown (Q<100) in
other applications a relatively high Q-factor (100≦Q<1000) may
be desired. The depth "D" of the holes 12 was chosen to provide the
required spectral separation between the excitation and extraction modes.
The thickness `t` of the TiO2 high index layer 26 was chosen to fine
tune the spectral location of the resonant modes. The photonic crystal
sensor of FIG. 1a was cost-effectively fabricated by a nano-replica
molding approach in an asymmetric configuration, so as to provide the
required mechanical stability and maintain a simplistic fabrication
procedure. A combination of high refractive index (RI) material (layer
26) and low refractive index material (layer 24, "Nanoglass"®,
Honeywell) was important in order to provide sufficient effective index
and positioning of the modes, respectively. Using a low RI material for
layer 24 allows the modes to be positioned closer to the device's upper
surface 11, whereas a high RI material for layer 24 which would draw the
modes deeper into the device 10 and subsequently reduce their interaction
with the fluorophores. The top surface 11 and cross section SEM images of
a fabricated model device are shown in FIG. 2B. The selection of
parameters of the device (refractive index, thickness, periodicity, etc.)
can be chosen to provide the desired location of resonant modes, as
further described herein.

[0058]While an array of holes is shown in the embodiment of FIG. 2a, other
types of periodic structures are possible. Such structures generally have
a configuration of periodic high and low regions (referred to herein
occasionally as "grooves"), which can take a variety of forms. In one
embodiment, the thickness of the high index layer 26 is in the range 30
to 1000 nm, e.g. 50 to 300 nm, preferably 50-200 nm. The period of the
corrugated structure may be in the range 200 to 1000 nm, e.g. 200 to 500
nm, preferably 250-500 nm. The ratio of the groove depth to the thickness
of the high index layer 26 lies in the range 0.02 to 1 e.g. 0.25 to 1,
preferably 0.3 to 0.7, and the ratio of the grooves width to the period
of the grooves ("duty-cycle") lies in the range 0.2 to 0.8, e.g. 0.4 to
0.6. Increasing the thickness of the high refractive index layer 26, the
refractive index of the layer 26, the refractive index of the low index
periodic grating layer 24, or the period of the grating will tend to
increase the wavelength of the resonant mode.

[0059]The grooves may be generally rectangular in cross-section.
Alternatively, the grooves may be sinusoidal or of saw tooth
cross-section. The surface structure may be generally symmetrical.
Preferred geometries include rectangular, sinusoidal and trapezoidal
cross-sections. Alternatively, the grooves may be of saw tooth
cross-section (blazed grating) or of other asymmetrical geometry. In
another aspect the groove depth may vary, e.g. in periodic modulations.

[0060]The support or platform may be square or rectangular and the grooves
may extend linearly along the platform so as to cover the surface.
Alternatively the platform may be disc shaped and the grooves may be
circular or linear.

[0061]The grating structure can take variety of one and two dimensional
forms, including two-level, two dimensional gratings, as disclosed in
published PCT application WO 2007/0179024, the contents of which are
incorporated by reference herein. These include square lattices and
hexagonal lattices that have symmetry in three directions along the
planar surface of the structure.

[0062]The corrugated, periodic grating surface may be optimized for one
particular excitation wavelength and for one particular type of
polarization. By appropriate means, e.g. superposition of several
periodic structures which are parallel or perpendicular one with another,
periodic surface relief can be obtained that are suitable for multiple
wavelength use of the photonic crystal sensor ("multicolor"
applications). Alternatively, individual sensing areas on one platform
may be optimized for different wavelengths and/or polarization
orientations.

[0063]In another embodiment, the photonic crystal is constructed so as to
exhibit a first extraction resonance mode in a first spatial area of the
photonic crystal and a second extraction resonance mode in a second
spatial area of the photonic crystal distinct from the first spatial
area. In other words, the construction of the surface grating structure
can vary spatially (in X and Y directions) such that different areas of
the photonic crystal exhibit different extraction resonant modes. This
spatial pattern of different extraction modes can be repeating. The first
extraction resonance mode has a spectrum at least partially overlapping
the emission spectrum of a first predetermined fluorophore (e.g., a
particular quantum dot) and wherein the second extraction resonance mode
has a spectrum at least partially overlapping the emission spectrum of a
second predetermined fluorophore different from the first predetermined
fluorophore (e.g., a second quantum dot).

[0064]Additionally, the construction of the surface grating structure 12
can vary spatially (in X and Y directions) such that different areas of
the photonic crystal exhibit different extraction resonant modes. As an
example, the photonic crystal exhibits different excitation resonant
modes at different spatial regions on the surface so that the fluorescent
dye Cy5 is excited in one location; and the fluorescent dye Cy3 is
excited in another nearby location. One could alternate between a group
of fluorophores (red, green, blue) much like how a single pixel of a
video display is comprised of multiple color emitters arranged close to
each other.

[0065]In another possible embodiment the photonic crystal is constructed
so as to exhibit a plurality of extraction resonance modes, each of the
plurality of extraction resonance modes having a resonant spectrum at
least partially overlapping an emission spectrum of a different
predetermined fluorophore.

[0066]Enhanced Excitation

[0067]When externally incident light 13 (FIG. 2a) interacts with
periodically modulated structures (holes 12) in the sub-wavelength regime
for a photonic crystal, only the 0th order forward and backward
diffracted waves can propagate. The periodicity however, also allows for
phase-matching of higher (evanescent) orders to localized leaky modes
supported by the photonic crystal 10. Once excited, the leaky modes,
defined by a complex propagation constant, possesses a finite lifetime as
they are leaked out both in the forward (transmitted) and backward
(specular) directions. The backward reradiated waves are in phase and
constructively interfere with the 0th backward diffracted order
while the forward reradiated waves are out of phase with the
0th forward diffracted order by π radians, causing
destructive interference and consequently resulting in zero transmission.
Thus, the external excitation of the leaky modes by means of incident
light 13 (FIG. 2a) is associated with a 100% reflection phenomenon for
the resonant wavelength, assuming a defect-free, lossless system. Since
the excited leaky modes are radiative but localized in space during their
finite lifetimes, they can be engineered to have very high energy density
within regions of the photonic crystal at resonance. The magnitude of
this energy density is directly related to the resonant mode lifetime or
Q-factor of resonance, which in turn can be controlled by adjusting the
device parameters (thickness, refractive index, depth of holes,
periodicity, etc.). Therefore, the intensity of emission of a fluorophore
(14, FIG. 1) (which is absorptive at the resonant wavelengths) can be
greatly enhanced by placing the fluorophores in proximity to regions
where the resonant modes concentrate most of their energy. In the example
of FIG. 2A, this region is the bottom of the holes, as shown in FIG. 4a,
as will be explained below.

[0068]Enhanced Extraction

[0069]Concurrently with the enhanced excitation as just described, the
existence of leaky modes in the photonic crystal 10 that overlap the
fluorescence emission spectrum opens up additional pathways for the
emitted light to escape into free-space. Besides direct emission, the
fluorescence can couple to the overlapping leaky modes and Bragg scatter
out of the photonic crystal sensor, thereby greatly reducing the amount
of light trapped as guided-modes, in comparison to an un-patterned
substrate (as explained above in FIG. 1). If the dispersion of these
overlapping emission leaky modes is close to the F-point band-edge, i.e.
K11 (magnitude of in-plane wave vector)˜0, a significant
amount of the emitted light will be extracted within small angles with
the vertical. It can thus be appreciated that enhancement of fluorescence
can be achieved by enhanced excitation and enhanced extraction acting in
concert together at the same time.

[0070]Results for Example 1

[0071]Studying the reflection/transmission properties of a photonic
crystal is a convenient technique to map out the dispersions of the leaky
modes. Rigorous Coupled-Wave Analysis (RCWA) techniques were used to
simulate how the device of FIG. 2a would respond in transmission to
externally incident radiation.

[0072]FIG. 3a shows the computed leaky mode band structure, i.e. spectral
location and transmission efficiency of the resonances as a function of
the angle of incidence (θ) of light 13 (FIG. 2a), along the
Γ-M direction. As θ is increased from 0°,
Ku.sub.∥ begins to increase and results in degenerate resonances
to separate into their respective constituent orders, as indicated at 40
in FIG. 3a. Experimental verification of the band structure of the
fabricated device was carried out by mounting the device in a linear
transmission setup, illuminating it with collimated white light and
plotting the resulting transmitted spectrum as a function of θ,
results of which are shown in FIG. 3(b). Excellent qualitative agreement
between simulation and experiment in the 460 nm 500 nm range was seen,
where the RIs of the materials vary slowly. Theoretically, it was
predicted that the resonance at λ=488 nm should occur at
θ=11.2° (the resonance indicated the region 42 in FIG. 3a),
and this is accurately observed in experiment. At shorter wavelengths,
the RI of TiO2 begins to increase divergently (n.sub.TiO2=2.7 at
λ=400 nm) and is considerably less at longer wavelengths
(n.sub.TiO2=2.36 at λ=600 nm), leading to deviations from
simulations assuming a constant RI (n.sub.TiO2=2.46 at λ=488 nm).
This is clearly seen in the theoretically predicted higher order bands
originating from shorter wavelength resonances and red-shifted longer
wavelength bands, which experimental results do not agree with. For the
excitation mode shown in FIG. 3A, the Q-factor of resonance was found to
be ˜155. The relatively high Q-factor is indicated in FIG. 3a by
the thin, highly defined line 41 of zero transmission efficiency in the
region 42.

[0073]FIGS. 4a and 4b show the simulated electric near-field intensity
(E2) (normalized to the unit amplitude incident field) at the two
available surfaces of the device, for the excitation of the resonant mode
at λ=488 nm. FIG. 4a shows the intensity at the bottom of the holes
12 of FIG. 2a. FIG. 4b shows the intensity at the upper surface 11 of the
sensor 10. The influence of the resonance phenomenon on the resulting
near-fields is clearly seen as manifested in the enhanced electric field
intensity. Similar enhancement can also be seen for the magnetic
near-fields. It is apparent that for the lower available surface (FIG.
4a) (bottom of the hole) the excitation mode concentrates its energy
within the cavity region (the term cavity is used here strictly in
relation to the shape of the cross-section). At the upper surface (FIG.
4b), the energy is concentrated at the cavity periphery and beyond. Both
the bottom of the well and the areas on the surface 11 adjacent to the
holes are where the fluorophore will be present during use, hence the
device exhibits strong intensities in the excitation resonance mode in
the areas of interest. Above the surfaces shown in FIGS. 4a and 4b, the
field intensity decays exponentially (as previously shown). In practice,
with a finite fluorophore which is not at the surface and unavoidable
losses, the exact near-field intensity available to the fluorophore will
always be lower than shown in FIGS. 4a and 4b.

[0074]The amount of amplification for enhanced excitation detection is
related to the power transferred from the device structure to a
distribution of fluorophores on the sensor surface at the excitation
wavelength of the fluorophore. The power density distribution of the
sensor surface at the resonant wavelength, provided that the resonant
wavelength is matched to the excitation wavelength, therefore provides a
means for comparing the sensitivity of different device designs. One can
define the cross product E (max)×H (max) as a field power or
"magnification factor". While a more thorough analysis of the intensity
distribution of the evanescent field from the tops, bottoms, and sides of
the structure, and a detailed integration of power density to account for
differences between higher and lower power regions would provide a more
exact prediction of whether one device will function more effectively
than another, the product of the maximum magnitude of an E component with
an orthogonal H component provides a very simple, rough way of comparing
designs. Nevertheless, studying the near-field intensity at the available
surfaces gives a convenient metric to optimize the photonic crystal
design. In this case, it is also important to mention that due to the
inherent asymmetry of the photonic crystal, the mode concentrates its
energy in the high index layer (26) and is biased more towards the
grating layer below it. By reversing the asymmetry (that is, by flooding
the device surface with higher index material, such as water) we can
reverse this biasing and to an extent, `draw` the resonance mode closer
to the device surface, therefore further increasing the mode interaction
with the fluorophore. Such a modification will be easily adaptable for
enhanced fluorescence biosensors, for example, where the analytes bound
to the fluorophores are typically in an aqueous buffer solution.
Near-field scanning optical microscopy (NSOM) images of the fabricated
devices excited close to the resonant wavelength have confirmed
enhancement and spatial localization of the electric field intensity.

[0075]The effect of the leaky modes that overlap the emission spectrum of
the fluorophores, which as per design, provide maximum overlap close to
the Γ-point band-edge, will now be discussed. Such a choice is
easily justified for maximal near-vertical extraction. In this enhanced
extraction phenomenon, the Q-factor of the extraction modes will
determine the rate at which coupled radiation will be scattered into
free-space. A low Q-facfor (implying a short mode lifetime) would be
beneficial in some applications, as the coupled radiation can be
scattered faster and thus the interaction of the radiation with losses in
the system can be limited. A low Q-factor is also desirable from the
standpoint that the radiation emitted from the fluorophores has a finite
bandwidth, and a broad leaky resonance can scatter more of the emitted
wavelengths in a given direction. Since the polarization and directions
of the emitted fluorescence for a fluorophore (e.g., quantum dot) in
free-space can be assumed to be arbitrary, the various available leaky
modes that can interact with the emitted light are considered. The
experimentally determined dispersion of the leaky modes supported by the
photonic crystal in the Γ-X and Γ-M directions and for
orthogonal polarizations (S and P) is shown in FIGS. 5a, 5b and 5c. (If
an electromagnetic wave is propagating toward a sensor surface at an
angle, it will have two orthogonal components of electric field. The "S"
component is the one with the electric field vector oriented parallel to
the sensor surface (so one can think of "S" standing for "skim", since
the electric field vector skims the surface), whereas the "P" component
is the electric field component with the vector oriented directly into
the sensor surface, and one can think of P as standing for "Plunge" as
the electric field vector plunges into the sensor surface. Incident light
can have both components at the same time.)

[0076]The case involving Γ-M and S polarization is already shown in
FIG. 3(a). It is clearly seen that the QD fluorophore whose emission
spectrum is (centered at λ=616 nm) can couple to leaky modes
supported by the photonic crystal and be extracted out of the device,
because the photonic crystal has a resonance mode indicated by the line
45 in FIGS. 5a and 5c which includes a region of resonance indicated at
47 which includes λ=616 nm at the angle of incidence 11.5 degree.
From experiment, the Q-factor for the extraction modes was ˜92.

[0077]To quantify the effects of the two fluorescence enhancement schemes,
the fabricated devices were cleaned using de-ionized water/isopropyl
alcohol, and the QDs (CdSe/ZnS core-shell type, Evident Technologies,
peak emission at λ=616 nm) were diluted in toluene and made up to a
concentration of 1.235 nM. The dilute solution of QDs was dropcast on and
off (to serve as a reference) the photonic crystal surface. After the
drying of the spots, the devices were scanned on a commercially available
laser scanner (LS 2000, Tecan), equipped with a 25 mW, 488 nm solid-state
laser and a photo multiplier tube (PMT) to record the fluorescence
signals. The scanner provides the ability to launch the incident
illumination at angles tunable from 0° to 25° in steps of
0.1°, along a single vertical axis and single polarization. In
order, to quantify the extraction enhancement and the excitation
enhancement effects, the devices were scanned at the resonant angle
(θ=11.2°) and a non-resonant angle (θ=0°). For
the sake of clarity, the resonant angle is defined as the launch angle
(θ) for which the leaky mode at λ=488 nm is excited. FIGS. 6a
and 6b show the scanned images taken at θ=11.2° and
θ=0° respectively. The circular regions 50 on the images
show the areas over which intensity information was averaged. Table 1
shows the raw data measured in PMT counts for the two cases over multiple
measurements. "PC" in Table 1 indicates "photonic crystal."

[0078]The enhancement (calculated by (S1-B1)/(S2-B2)) for
θ=11.2° was 108.89±4.09, and for θ=0° was
13.21±0.26. The observed enhancement at θ=0° is
attributed mainly to the enhanced extraction provided by the photonic
crystal. Since the extraction effect is only related to the dispersive
properties of the photonic crystal, it should not be affected by a change
in the launch angle of the incident light. Using this assumption, the
total enhancement obtained for the resonant angle is divided by the
enhancement obtained for the non-resonant angle and a value of
8.24±0.36 times for fluorescence enhancement by the near-fields was
obtained.

[0079]Discussion

[0080]The result for fluorescence enhancement due to the enhanced
near-fields, at first glance, is much lesser than the peak intensity of
the near-fields shown in FIGS. 4a and 4b. However, one could expect that
the spatially-averaged near-field enhancement would be much lower due to
the specific pattern of the field distributions, and more important in
deciding the resulting enhancement due to the nonspecific positioning of
the QDs. Furthermore, it was found that due to the inherent absorption
and fluorescence at visible wavelengths of most materials used in such
fabrication processes results in additional loss to the resonance. In the
fabricated device, the combination of the spin-on glass material
(Nanoglass®, Honeywell) for the grating layer and TiO2 for the
high index layer was strongly absorbing and fluorescent at the excitation
wavelength. This can be seen by the enhancement of the background signal
from regions on the photonic crystal where no quantum dots are present
(FIG. 6(b)). Indeed, the enhanced fields produced due to the resonance
effect serve also to boost the background signal over 13 times,
presenting a strong, undesirable loss mechanism that reduces the
excitation intensity available to the QDs. Alternative fabrication
methods and material choices can be used to minimize such losses, as
known in the art.

[0081]For the enhanced extraction case, the fluorescence enhancement is
believed to be mostly related to Bragg scattering. The structure
fabricated in this example, due to its asymmetric nature, cannot posses a
bandgap for either TE or TM-like modes and therefore, the effect of
inhibited spontaneous emission into undetectable waveguide modes is
absent. Time-resolved fluorescence measurements on the QDs both on and
off the photonic crystal surface helped verify the absence of cavity
enhanced spontaneous emission via the Purcell effect (data not shown).
Enhanced extraction was verified by angle-resolved fluorescence
measurements. By illuminating the photonic crystal and measuring the
fluorescence emitted by the QDs at different angles, the enhanced
extraction phenomenon was verified and shows strong coupling between the
extraction modes and the QDs. The extraction effects may be further
optimized by reducing the anisotropy of the in-plane wave vector, by
employing photonic lattices whose Brillouin zones are more circular, e.g.
triangular or quasi-periodic lattices. The density of extraction modes
will also affect the extraction efficiency. A greater density of modes
that overlap the emission spectrum of the QDs, would result in stronger
scattering and consequently extraction effects. Finally, by engineering
the spectral overlap and dispersion properties of the various leaky modes
supported by the photonic crystal one can extend the enhancement effect
to a wide range of fluorescent species.

[0082]Such a fluorescence enhancement scheme can be invaluable to the
application of fluorescent biosensing using QDs, for example. Given the
excellent applicability of QDs to serve as fluorescent probes, a highly
sensitive fluorescence detection system is provided that will enable
working at very low/single molecule analyte concentrations. Such a
detection scheme will inherently incorporate low background fluorescence,
as the QD tags close to the biosensor surface will experience maximum
fluorescence enhancement, similar to total internal reflection
fluorescence (TIRF) microscopy.

[0083]Here, we have demonstrated resonant enhancement of over 108 times in
fluorescence from QDs on the surface of a 2D photonic crystal. This has
been achieved by engineering the photonic crystal such that it possesses
leaky eigenmodes (resonance mode) at the absorption and emission
wavelengths of the QDs. The results of this work can be adapted to a wide
variety of optical applications involving QDs, including high brightness
LEDs, optical switches and high sensitivity biosensors.

[0084]Fabrication Methods

[0085]The two-dimensional photonic crystals described herein can be
fabricated by a nano-replica molding process, described in the previously
cited patent literature. Briefly, electron beam lithography (JEOL
JBX-6000FS) was used to define a two-dimensional `square lattice of
holes` surface structure of period Λ=300 nm and hole radius r=90
nm on a SiO2/Si substrate with PMMA as the mask layer. The pattern
was exposed to a size of 3×3 mm2 followed by development and
dry etching in a CHF3 reactive ion etching process. The resulting surface
structure was subsequently transferred to a glass substrate (PET film or
glass), coated with a low-index porous spin-on-glass (Nanoglass,
Honeywell) using an intermediate polydimethylsiloxane (PDMS) stamp. A
thin layer of high index TiO2 (t=125 nm) was then sputtered (AJA
International Inc.) to form the final device. The refractive indices (RI)
of the Nanoglass (ng) and TiO2 materials as determined by
spectroscopic ellipsometry (Woolam) were nng=1.17 and
n.sub.TiO2=2.46 respectively at λ=488 nm.

[0086]Simulation and Device Design

[0087]A commercial implementation of the RCWA code (GSolver) was employed
for all the simulations. One period of the device was simulated, with
periodic boundary conditions applied to the x and y extents. The incident
radiation was set to be S-polarized plane waves incident from above the
device and along the Γ-M direction (θ=45°, the choice
of these launch parameters were essentially dictated by limitations of
our experimental setup). To improve the calculation speed for the leaky
mode band structure, the materials were assumed lossless and the RI
dispersion was assumed to be flat about λ=488 nm. Near-field
calculations however, were performed including the complex component of
the material refractive indices (kNG=0 and k.sub.TiO2=0.00036) and
retaining 12 harmonics in both the x and y directions.

[0089]Photonic crystal (PC) slab devices fabricated by nano-replica
molding were inspected using the Witec Alpha near-field scanning
microscope (NSOM). The devices were excited with λ=488 nm
excitation from an argon-ion laser and the near fields were probed close
to resonance. The resulting near-field intensity map is shown in FIG. 7
and shows a distribution qualitatively identical to the simulated
near-field in FIG. 4(b), within the limited lateral resolution of the
NSOM.

[0090]Enhancement of the near-field is also evident in the NSOM image of
FIG. 7. Defects incorporated into the device during fabrication are
clearly visible as regions where the nearfield distributions are
distorted.

[0091]From FIGS. 4a and 4b one can predict the absolute maximum
enhancement obtainable by spatially averaging the predicted near-field
distributions over the device surface. For the distributions relevant to
the designed devices, a maximum average intensity of ˜240 times is
calculated at the device surface. However due to unavoidable resonator
losses and finite size of the quantum dots (˜5 nm), the practical
enhancement in the excitation intensity would be lower.

[0092]Experiments verifying the enhanced extraction provided by the
photonic crystal.

[0093]In order to verify the enhanced extraction effect of the photonic
crystal due to the overlap of its leaky eigenmodes with the fluorescence
spectrum of the QDs, angle-resolved fluorescence measurements were
performed. The QDs were used at a 100× higher concentration (123.5
nM) for this experiment to provide sufficient signal for detection. Using
a higher concentration resulted in the band structure of the leaky modes
being slightly red-shifted, due to the increase in effective-index for
the resonances. Consequently, this results in slightly increased angles
for extraction.

[0094]The enhanced extraction phenomenon is believed to occur when the
leaky modes (termed extraction modes) of the photonic crystal overlap
with the QD fluorescence emission spectra. This overlap creates
`channels` into which the QDs can couple their energy. Since the modes
are by definition leaky, this coupled emission from the QDs must also
leak into free-space. The direction (angles) of leakage will also follow
the dispersion of the extraction modes. In order to extract all the
emitted light in a single direction, the bandwidth of the leaky mode to
should be equal to or larger than the fluorescence bandwidth. This has
been explained as the rationale behind designing photonic crystals with a
relatively low Q-factor extraction modes.

[0095]In experiment, the photonic crystal containing the QDs on its
surface was mounted on a fixed stage. Incident light from a λ=488
nm, 10 mW argon-ion laser was normally incident on the device, and
provided the required excitation for the QDs. It must be noted that the
incident light is not resonant with the photonic crystal at this angle,
and therefore the near-fields are not enhanced. The fluorescence was
detected from the sample by a fiber probe set at a distance of L=10 cm
from the device center. A band stop filter filtered out the laser
excitation so that only fluorescence from the QDs was observed. The probe
was rotated about the device and the fluorescence spectrum was collected.
By rotating the sample orientation, the spectrum Was recorded for both
the Γ-M and Γ-X directions.

[0096]FIG. 8 shows the angle resolved fluorescence spectrum as measured
from the PC when the measurement is taken along the Γ-M direction.
Since a polarizer is not used to filter the emitted radiation, all
polarizations of emitted radiation are detected in the same measurement.
For the case of the Γ-M direction, a broad angle-independent
fluorescence feature that ranges from 600-650 nm is seen, indicated by
the lighter region 100. This is the fluorescence from the QDs.
Superimposed upon this feature, strong features that match the extraction
mode band structure experimentally determined in FIGS. 2 & 4 are seen,
indicated by the bright regions of highest detected intensity in FIG. 8,
indicated at 102. This is a clear representation of the strong coupling
between the QD fluorescence and the leaky modes of the PC. As seen, the
fluorescence is also enhanced when the leaky modes overlap the
fluorescence spectrum, the strongest enhancement being obtained when the
peak of the leaky mode overlaps the peak wavelength of the fluorescence
emission spectrum.

[0097]A different way of looking at the two-dimensional experimental data
shown in FIG. 8 is shown in FIG. 9, where slices of data from FIG. 8 are
normalized and superimposed upon each other. In FIG. 8, the curve 104
represents the emission of the quantum dots when no photonic crystal is
present. The curve 106 represents the condition when the leaky mode just
begins to overlap the fluorescence spectrum of the quantum dots (at
normal incidence, θ=0°). A clear modification in spectral
characteristics is seen, as in the appearance of a lower wavelength peak
108 resulting from enhancement of the QD sideband emission. When the peak
of the emission matches the peak of the quantum dot emission
(A=10°, dark square curve), a dramatic change in spectral
characteristics of the emitted radiation involving emission bandwidth
reducing to roughly half its original value is evident, indicated by
curve 110. Thus, the presence of the photonic crystal results in strong
spectral and spatial modification of the fluorescence emitted by the QDs.

[0098]FIG. 10 shows the measurement of angle-resolved fluorescence for the
Γ-X direction. In this case, two extraction bands 120 and 122
overlapping the fluorescence spectrum 100 from the QDs are seen in
comparison to one as shown in FIG. 8. This is due to the fact that in the
Γ-X direction, the leaky band structure for the extraction modes is
different for different polarizations of coupled light. Each of the bands
120 and 122 appearing in FIG. 10 corresponds to either S-polarized or
P-polarized light being emitted by the QDs. In the Γ-M direction,
the dispersion of the extraction leaky modes is independent of the
polarization. This is also seen clearly in FIGS. 3 & 5, where the
dispersions of the extraction modes for the Γ-M direction are same
for both polarizations (S & P) but different for different polarizations
in the Γ-X direction.

[0099]FIG. 11 is a graph of the intensity of quantum dot emission as a
function of time, showing that quantum dots decay significantly faster
when placed on either a TiO2 substrate or a photonic crystal as compared
to a glass slide.

[0100]While the above example has used a quantum dot fluorophore, the
invention is applicable to detection of any fluorophore or
fluorescently-labeled group. Using a non-QD fluorophore (such as an
organic fluorescent dye) with the inventive photonic crystal sensor will
still provide significant fluorescence sensitivity enhancement.

[0101]Extraction Mode Only Sensors

[0102]While the above example has demonstrated a photonic crystal sensor
with resonance modes which overlap both the excitation and emission
spectra of a fluorophore, in another embodiment the photonic crystal can
be structured and arranged such that the photonic crystal exhibits a
resonant mode when illuminated with light at an incident angle θ
which at least partially overlaps the emission spectrum of the
fluorophore, but the photonic crystal does not simultaneously have a
resonance mode which overlap the excitation spectrum of the fluorophore.
Such a sensor would exhibit the enhanced extraction effect but not the
enhanced excitation effect. The sensor would be useful of many
applications, such as those described previously.

[0103]A sample testing system for testing a sample is envisioned using a
sensor featuring just the enhanced extraction mode. The system would
include a detection instrument comprising a light source and a detector,
and a photonic crystal sensor comprising a periodic grating structure,
with the sample including the fluorophore being placed on the periodic
grating structure. The light source of the detection system is oriented
relative to the photonic crystal sensor such that the light source
illuminates the photonic crystal sensor at a incident angle θ in
which the photonic crystal exhibits a resonant mode having a resonant
spectrum which at least partially overlaps the emission spectrum of the
fluorophore. The detector operates to detect radiation from the
fluorophore in the emission spectrum.

[0104]Yet further, in this embodiment, a method is provided for testing a
sample having a fluorophore bound to the sample. The method includes the
steps of placing the sample onto the surface of a photonic crystal
sensor; illuminating the photonic crystal biosensor with light at an
angle of incidence θ, the biosensor responsively exhibiting an
extraction resonance mode having a spectrum which at least partially
overlaps the emission spectrum of the fluorophore, the illumination and
the resulting extraction resonance mode causing the fluorophore to emit
light; and collecting the emitted light from the fluorophore and
directing the emitted light onto a detector.

Other Examples

Photonic Crystal Constructions

[0105]The substrate layer and grating layers of the photonic crystal
sensor may be formed from inorganic materials such as glass, SiO2,
quartz, silicon, and of different organic and inorganic components or
layers as composite materials. Alternatively the layers can be formed
from organic materials such as polymers preferably polycarbonate (PC),
poly (methyl methacrylate) (PMMA), polyimide (PI), polystyrene (PS),
polyethylene (PE), polyethylene terepthalate (PET) or polyurethane (PU).
Substrate materials also include polycarbonate or cyclo-olefin polymers
such as Zeanor®.

[0106]The high index of refraction layer on the top of the substrate may
be formed from inorganic materials. Examples include metal oxides such as
Ta2O5, TiO2, Nb2O5, ZrO2, ZnO or HfO2.

[0107]The embodiment of a two-dimensional grating structure suitable for
simultaneous fluorescence enhancement by enhanced fluorescence excitation
and enhanced extraction is disclosed and may be preferred in some
implementations. A two-dimensional grating can look like a waffle
(holes), a waffle iron (posts), or a chessboard configuration with
alternating high and low regions in two dimensions, for example.
Two-dimensional gratings can have different periods in the X and Y
directions. These features may have various profiles in the Z direction
such as angled or curved sidewalls. Thus, in the case of the waffle
pattern, the impressions or wells may have a rectangular rather than a
square shape. This added flexibility provided by two dimensional gratings
allows one to tune the resonance positions for enhanced excitation and
extraction detection to occur at different wavelengths. As an example,
the X periodicity can provide a sharp resonance at or near normal
incidence with wavelength tuned to excite the fluorophore while the Y
periodicity can yield a broad resonance that coincides with the emission
wavelength of the fluorophore. In one particular example, the X
periodicity provides a resonance tuned to excite a Cy3 fluorophore with
green light, while the Y periodicity gives a broad resonance that
coincides with the emission wavelength of Cy3.

[0108]A single photonic crystal surface may be used to support, in
parallel, a large number of fluorescence assays in the form of an array
of probes or capture molecules that are deposited upon different
locations. Each probe/capture molecule (the terms probes and capture
molecules are used interchangeably herein) may contain individual and/or
mixtures of capture molecules which are capable of affinity reactions.
The shape of an individual capture molecule may be rectangular, circular,
ellipsoidal, or any other shape. The area of an individual capture
element may be any suitable area, such as between 1 μm2 and 10
mm2, between 20 μm2 and 1 mm2 and in one embodiment,
between 100 μm2 and 1 mm2. The capture molecules may be
arranged in a regular two dimensional array. The center-to-center (ctc)
distance of the capture elements may be any suitable distance, such as
between 1 μm and 1 mm, between 5 μm to 1 mm, and between 10 μm
to 1 mm.

[0109]The number of capture elements per sensing region is between 1 and
1,000,000, preferably between 1 and 100,000. In another aspect, the
number of capture elements to be immobilized on the platform may not be
limited and may correspond to the number of desired features under
investigation e.g. the number of genes, DNA sequences, DNA motifs, DNA
micro satellites, single nucleotide polymorphisms (SNPs), proteins or
cell fragments constituting a genome of a species or organism of
interest, or a selection or combination thereof. In a further aspect, the
platform of this invention may contain the genomes of two or more
species, e.g. mouse and rat, or human and mouse.

[0110]Sensor Platforms

[0111]The photonic crystal structures of this disclosure may be produced
uniformly over large surface areas using a nanoreplica molding process.
After manufacture, the structure may be incorporated onto the surface of
microscope slides, within standard format microplates, or any other
convenient assay format, including microarray formats. A microarray
format typically includes a large number (e.g., 10,000, or 100,000) of
distinct locations. Such locations are typically laid out in a regular
grid pattern in x-y coordinates. However, a microarray can be laid out in
any type of regular or irregular pattern. For example, distinct locations
can define a microarray of spots of one or more specific binding
substances. A microarray spot can be about 50 to about 500 μm in
diameter or any other suitable diameter. A microarray on a support to be
used in this invention can be used by placing microdroplets of a sample
including one or more specific binding substances and fluorophores onto,
for example, an xy grid of locations on a two-dimensional grating or
cover layer surface. When the biosensor is exposed to a test sample
comprising one or more binding partners, the binding partners will be
preferentially attracted to distinct locations on the microarray that
comprise specific binding substances that have high affinity for the
binding partners. Some of the distinct locations will gather binding
partners onto their surface, while other locations will not.

[0112]One example of a microarray to be used in a method according to the
present invention is a nucleic acid microarray, in which each distinct
location within the array contains a different nucleic acid molecule. In
this embodiment, the spots within the nucleic acid microarray detect
complementary chemical binding with an opposing strand of a nucleic acid
in a test sample.

[0113]The sensors described here can be used to sensitively analyze a
variety of analytes. Some examples of analytes that can be detected using
the sensors and methods herein include, but are not limited to, one or
more: proteins, peptides, DNA molecules, RNA molecules, oligonucleotides,
lipids, carbohydrates, polysaccharides; glycoproteins, lipoproteins,
sugars, cells, bacteria, viruses, candidate molecules and all
derivatives, variants and complexes of these, which have a fluorescent
label. Other fluorescent substances can be detected, as known in the art.
Nanomaterials such as quantum dots or functionalized quantum dots may be
used. Applications include gene expression microassays where genes may be
detected at lower expression levels and/or with Y smaller sample volumes.
Other applications include protein detection assays, such as detection of
protein biomarkers in bodily fluids for disease diagnostic tests, where
proteins are present at very low concentration. Detection by the method
described in this invention would be more sensitive than commonly used
ELISA assays, but with a simpler assay protocol. In addition, fluorescent
imaging of cells and proteins using fluorescent microscopes can utilize
the techniques presented here, where the improved sensitivity can be used
to observe dye molecules at lower concentrations and/or to use lower-cost
imaging came due to improved signal-to-noise ratio

[0114]Alternative Grating Structures

[0115]In one embodiment, a support to be used in a method of the invention
will be illuminated with white light that will contain light of every
polarization angle. The orientation of the polarization angle with
respect to repeating features in a biosensor grating will determine the
resonance wavelength. For example, a "linear grating" biosensor structure
consisting of a set of repeating lines and spaces will have two optical
polarizations that can generate separate resonant reflections. Light that
is polarized perpendicularly to the lines is called "s-polarized," while
light that is polarized parallel to the lines is called "p-polarized."
Both the s and p components of incident light exist simultaneously in an
unfiltered illumination beam, and each generates a separate resonant
signal. A support structure can generally be designed to optimize the
properties of only one polarization (the s-polarization), and the
non-optimized polarization is easily removed by a polarizing filter.

[0116]In order to remove the polarization dependence, so that every
polarization angle generates the same resonant reflection spectra, an
alternate structure can be used that consists of a set of concentric
rings. In this structure, the difference between the inside diameter and
the outside diameter of each concentric ring is equal to about one-half
of a grating period. Each successive ring has an inside diameter that is
about one grating period greater than the inside diameter of the previous
ring. The concentric ring pattern extends to cover a single sensor
location--such as a microarray spot or a microtitre plate well. Each
separate microarray spot or microtitre plate well has a separate
concentric ring pattern centered within it. All polarization directions
of such a structure have the same cross-sectional profile. The concentric
ring structure must be illuminated precisely on-center to preserve
polarization independence. The grating period of a concentric ring
structure is less than the wavelength of the resonantly reflected light.
The grating period is about 0.01 micron to about 1 micron, in one
embodiment. The grating depth is about 0.01 to about 1 micron, in one
embodiment.

[0117]In another embodiment, an array of holes or posts are arranged to
closely approximate the concentric circle structure described above
without requiring the illumination beam to be centered upon any
particular location of the grid. Such an array pattern is automatically
generated by the optical interference of three laser beams incident on a
surface from three directions at equal angles. In this pattern, the holes
(or posts) are centered upon the corners of an array of closely packed
hexagons. The holes or posts also occur in the centre of each hexagon.
Such a hexagonal grid of holes or posts has three polarization directions
that "see" the same cross-sectional profile. The hexagonal grid
structure, therefore, provides equivalent resonant reflection spectra
using light of any polarization angle. Thus, no polarizing filter is
required to remove unwanted reflected signal components. The period of
the holes or posts can be about 0.01 μm to about 1 μm and the depth
or height can be about 0.01 μm to about 1 μm.

[0118]The detecting system may be arranged to detect luminescence such as
fluorescence. Affinity partners can be labeled in such a way that Forster
fluorescence energy transfer (FRET) can occur upon binding of analyte
molecules to capture molecules. The maximum of the luminesce labels can
be used to modify capture elements, assayed molecules in the analyte, or
any other species, e.g. endogeneous/exogeneous controls, spacer
molecules, primers, bio/materials, that interact with the sensor surface.

[0119]The luminescence dyes used as markers may be chemically or
physically, for instance electrostatically, bonded to one or multiple
affinity binding partners (or derivatives thereof) present in the analyte
solution and/or attached to the platform. In case of naturally occurring
oligomers or polymers such as DNA, RNA, saccharides, proteins, or
peptides, as well as synthetic oligomers or polymers, involved in the
affinity reaction, intercalating dyes are also suitable. Luminophores may
be attached to affinity partners present in the analyte solution via
biological interaction such as biotin/avidin binding or metal complex
formation such as HIStag coupling.

[0120]One or multiple luminescence markers may be attached to affinity
partners present in the analyte solution, to capture elements immobilized
on the platform, or both to affinity partners present in analyte solution
and capture elements immobilized at the platform, in order to
quantitatively determine the presence of one or multiple affinity binding
partners.

[0124]While the above examples have used quantum dots as the fluorescent
molecule which is excited by incident radiation, other fluorophores can
be used in accordance with the inventive biosensor.

[0125]Transfluorospheres or derivatives thereof may be used for
fluorescence labeling, and chemiluminescent or electroluminescent
molecules may be used as markers.

[0126]Luminescent compounds having luminescence in the range of from 400
nm to 1200 nm which are functionalised or modified in order to be
attached to one or more of the affinity partners may be used, including
derivatives of: polyphenyl and heteroaromatic compounds, stilbenes,
coumarines, xanthene dyes, methine dyes, oxazine dyes, rhodamines,
fluoresceins, coumarines, stilbenes, pyrenes, perylenes, cyanines,
oxacyanines, phthalocyanines, porphyrines, naphthalopcyanines, azobenzene
derivatives, distyryl biphenyls, transition metal complexes e.g.
polypyridyl/ruthenium complexes, tris (2,2' bipyridyl) ruthenium
chloride, tris(1,10-phenanthroline) ruthenium chloride, tris (4,7
diphenyl-1,10-phenanthroline) ruthenium chloride and
polypyridyl/phenazine/ruthenium complexes, such as
octaethyl-platinum-porphyrin, Europium and Terbium complexes may be used
as luminescence markers, nanoparticles, microparticles, or any other
light emitting species that can be excited by evanescent fields.

[0127]Suitable for analysis of blood or serum are dyes having absorption
and emission wavelength in the range from 400 nm to 1000 nm. Furthermore
luminophores suitable for two and three photon excitation can be used.

[0128]Dyes which are suitable in this invention may contain functional
groups for covalent bonding, e.g. fluorescein derivatives such as
fluorescein isothiocyanate. Also suitable are the functional fluorescent
dyes commercially available from Amersham Life Science, Inc., Texas, and
Molecular Probes Inc. Other suitable dyes include dyes modified with
deoxynucleotide triphosphate (dNTP) which can be enzymatically
incorporated into RNA or DNA strands. Further suitable dyes include
Quantum Dot Particles or Beads (Quantum Dot Cooperation, Palo Alto,
Calif.) or derivatives thereof or derivatives of transition metal
complexes which may be excited at one and the same defined wavelength,
and derivatives show luminescence emission at distinguishable
wavelengths.

[0129]Analytes may be detected either via directly bonded luminescence
markers, or indirectly by competition with added luminescence marked
species, or by concentration, distance, pH, potential-or redox
potential-dependent interaction of luminescence donors and
luminescence/electron acceptors used as markers bonded to one and/or
multiple analyte species and/or capture elements. The luminescence of the
donor and/or the luminescence of the quencher can be measured for the
quantification of the analytes.

[0130]In the same manner affinity partners can be labeled in such a way
that electron transfer or photoinduced electron transfer leads to
quenching of fluorescence upon binding of analyte molecules to capture
molecules.

[0131]Luminescent labels can be used to modify capture elements, assayed
molecules in the analyte, or any other species, e.g.
endogeneous/exogeneous controls, spacer molecules, primers,
bio/materials, that interact with the sensor surface.

[0132]The luminescence dyes used as markers may be chemically or
physically, for instance electrostatically, bonded to one or multiple
affinity binding partners (or derivatives thereof) present in the analyte
solution and/or attached to the platform. In case of naturally occurring
oligomers or polymers such as DNA, RNA, saccharides, proteins, or
peptides, as well as synthetic oligomers or polymers, involved in the
affinity reaction, intercalating dyes are also suitable. Luminophores may
be attached to affinity partners present in the analyte solution via
biological interaction such as biotin/avidin binding or metal complex
formation such as HIStag coupling.

[0133]One or multiple luminescence markers may be attached to affinity
partners present in the analyte solution, to capture elements immobilized
on the platform, or both to affinity partners present in analyte solution
and capture elements immobilized at the platform, in order to
quantitatively determine the presence of one or multiple affinity binding
partners.

[0137]The biosensor surface may include an adhesion promoting layer
disposed at the surface of the optically transparent layer (high index of
refraction layer) in order to enable immobilization of capture molecules.
The adhesion promoting layer may also comprise a microporous layer (for
example, ceramics, glass, Si) for further increasing assay and detection
efficacy or of gel layers which either can be used as medium for carrying
out the capture element immobilization and sample analysis, thereby
further increasing the assay and detection efficacy, or which allow
separation of analyte mixtures in the sense of gel electrophoresis. The
platform may be formed with a plurality of sensing areas or regions, each
having its own diffractive grooves.

[0138]In other words, immobilization of one or more probes/capture
molecules onto a biosensor surface can be performed so that a specific
binding substance will not be washed away by rinsing procedures, and so
that its binding to binding partners in a test sample is unimpeded by the
biosensor surface. Several different types of surface chemistry
strategies have been implemented for covalent attachment of specific
binding substances to, for example, glass for use in various types of
microarrays and biosensors. These same methods can be readily adapted to
a biosensor of the invention. Surface preparation of a biosensor so that
it contains the correct functional groups for binding one or more
specific binding substances is an integral part of the biosensor
manufacturing process.

[0139]One or more specific binding substances can hence be attached to a
biosensor surface by physical adsorption (i.e., without the use of
chemical linkers) or by chemical binding (i.e., with the use of chemical
linkers). Chemical binding can generate stronger attachment of specific
binding substances on biosensor surface and provide defined orientation
and conformation of the surface-bound molecules. For instance, some types
of chemical binding include, for example, amine activation, aldehyde
activation, and nickel activation. These surfaces can be used to attach
several different types of chemical linkers to a biosensor surface. While
an amine surface can be used to attach several types of linker molecules,
an aldehyde surface can be used to bind proteins directly, without an
additional linker. A nickel surface can be used to bind molecules that
have an incorporated histidine ("his") tag. Detection of "his-tagged"
molecules with a nickel-activated surface is well known in the art
(Whitesides, Anal. Chem. 68, 490 (1996)).

[0140]Immobilization of specific binding substances to plastic, epoxy, or
high refractive index material can be performed similarly to that
described for immobilization to glass. However, the acid wash step can be
eliminated where such a treatment would damage the material to which the
specific binding substances are immobilized. This is well known in the
art.

[0141]For the detection of binding partners at concentrations less than
about 0.1 ng/ml, it is possible to amplify and transduce binding partners
bound to a biosensor into an additional layer on the biosensor surface.
The increased mass deposited on the biosensor can be easily detected as a
consequence of increased optical path length. By incorporating greater
mass onto a biosensor surface, the optical density of binding partners on
the surface is also increased, thus rendering a greater resonant
wavelength shift than would occur without the added mass. The addition of
mass can be accomplished, for example, enzymatically, through a
"sandwich" assay, or by direct application of mass to the biosensor
surface in the form of appropriately conjugated beads or polymers of
various size and composition. This principle has been exploited for other
types of optical biosensors to demonstrate sensitivity increases over
1500 beyond sensitivity limits achieved without mass amplification. See,
e.g., Jenison et al., "Interference-based detection of nucleic acid
targets on optically coated silicon," Nature Biotechnology 19: 6265
(2001).

[0142]As an example, a NH2-activated biosensor surface can have a
specific binding substance comprising a single-strand DNA capture probe
immobilized on the surface.

[0143]The capture probe interacts selectively with its complementary
target binding partner. The binding partner, in turn, can be designed to
include a sequence or tag that will bind a "detector" molecule. A
detector molecule can contain, for example, a linker to horseradish
peroxidase (HRP) that, when exposed to the correct enzyme, will
selectively deposit additional material on the biosensor only where the
detector molecule is present. Such a procedure can add, for example, 300
angstroms of detectable biomaterial to the biosensor within a few
minutes.

[0144]A "sandwich" approach can also be used to enhance detection
sensitivity. In this approach, a large molecular weight molecule can be
used to amplify the presence of a low molecular weight molecule. For
example, a binding partner with a molecular weight of, for example, about
0.1 kDa to about 20 kDa, can be tagged with, for example,
succinimidyl6[amethyla(2pyridyldithio) toluamido] hexanoate (SMPT), or
dimethylpimelimidate (DMP), histidine, or a biotin molecule.

[0145]Detection Apparatus

[0146]The photonic crystal biosensors of this disclosure are used in
conjunction with an appropriate detection apparatus or instrument. The
particular nature and construction of the detection apparatus is not
especially important. The detection apparatus detects the luminescent
response of the fluorophores bound to a sample when the sample and
fluorophore are deposited on the surface of the biosensor and the sensor
is illuminated with light at a frequency which overlaps the excitation
spectrum of the fluorophore. Examples of appropriate detectors for
luminescence include CCD-cameras, photomultiplier tubes, avalanche
photodiodes, photodiodes, hybrid photomultiplier tubes, or arrays
thereof. The disclosure of the detection apparatus described in U.S.
patent application publications U.S. 2003/0027327; 2002/0127565,
2003/0059855 and 2003/0032039, U.S. Pat. Nos. 7,023,544, 7,064,844, and
published PCT application WO 2007/0179024, the contents of each of which
is hereby incorporated herein by reference. Since the detection apparatus
is described in the literature, a further explanation is omitted here for
the sake of brevity. The detection apparatus can be arranged to detect in
addition changes in the refractive index due to the coupling of the
sample and fluorophore to the sensor surface and resulting shift in the
peak wavelength of reflected light. The incident beam may be arranged to
illuminate the sensing area or all sensing areas on one common platform.
Alternatively the beam can be arranged to illuminate only a small subarea
of the sensing area to be analyzed and the beam and/or the platform may
be arranged so that they can undergo relative movement in order to scan
the sensing area of the platform. Accordingly, the detecting apparatus
may be arranged in an appropriate way to acquire the luminescence signal
intensities of the entire sensing area in a single exposure step.
Alternatively the detection and/or excitation means may be arranged in
order to scan the sensing areas stepwise.

[0147]The detection instrument includes a light generating unit which
illuminates the photonic crystal sensor. The light generating unit may
comprise a laser emitting a coherent laser beam. Other suitable light
sources include discharge lamps or low pressure lamps, e.g. Hg or Xe,
where the emitted spectral lines have sufficient coherence length, and
light-emitting diodes (LED). The apparatus may also include optical
elements for directing the laser beam so that it is incident on the
platform at an angle θ, and elements for shaping the plane of
polarization of the coherent beam, e.g. adapted to transmit linearly
polarized light.

[0148]Examples of lasers that may be used are gas lasers, solid state
lasers, dye lasers, semiconductor lasers. If necessary, the emission
wavelength can be doubled by means of nonlinear optical elements.
Especially suitable lasers are argon ion lasers, krypton ion lasers,
argon/krypton ion lasers, and helium/neon lasers which emit at
wavelengths between 275 and 753 nm. Very suitable are diode lasers or
frequency doubled diode lasers of semiconductor material which have small
dimensions and low power consumption.

[0149]Another appropriate type of excitation makes use of VCSEL's
(vertical cavity surface emitting lasers) which may individually excite
the recognition elements on the platform.

[0150]In an embodiment in which the photonic crystal is incorporated onto
a microscope slide, the detection instrument may include a microscope for
viewing the slide. The microscope may direct a magnified image of the
field of view onto an imaging device such as a charge coupled device
camera which then captures and stores images of the field of view.
Workstations incorporating microscopes and cameras are described in the
patent literature and therefore a detailed discussion of the features of
such as system are omitted for the sake of brevity.

[0151]FIG. 12 is block diagram of one possible embodiment of a detection
instrument 190 for use with photonic crystal sensor 10 featuring enhanced
excitation and enhanced extraction. The instrument 190 features a
modified upright fluorescence microscope 195. The instrument 190 includes
a HeNe laser 200 which directs light 260 through a neutral density filter
202 and a beam expander 204. The expanded beam is directed through an
aperture 206 and a 1/2 wave plate 208 for polarization and onto a
motorized stage of the microscope 195. The motorized stage includes a
linear stage 212 for travel in the X and Y directions. An adjustable
angular stage 210 is mounted to the linear stage 212 and is used to
adjust the beam angle θ. Laser light from the stage is directed via
a mirror 209 upwards to a manual stage 214 of the microscope 195. The
photonic crystal sensor 10 (e.g., incorporated into a microscope slide)
is placed on the manual stage 214. The laser excitation light 260 is
tuned to match the excitation band of a fluorophore present in the sample
placed on the sensor 10 and causes the fluorophore to emit fluorescence
(shown by line 250).

[0152]The combination of components 200, 202, 204, 206, 208, 210 and 212
provide for an angle-tunable, beam-expanded excitation laser input. The
laser beam 260 is expanded in the beam expander 204 to ensure uniform
excitation across the sensor 10 and maximum collimation, and cropped with
an aperture 206 to prevent photobleaching outside the imaging area.
Alignment of the laser beam to the sensor 10 is achieved using a set of
mirrors (one which is mounted on a precision linear stage 212),
polarization is adjusted with a half-wave plate 208, and attenuated with
a continuously variable neutral density filter 202. The beam is then
reflected off a gimbal-mounted mirror 209 and incident upon the sensor
10. The gimbal mount is controlled with a high precision motor and is
itself mounted on a linear stage 212 that also employs a high-precision
motorized drive. This linear stage 212 ensures the illumination area on
the sensor 10 remains fixed as the excitation beam angle θ is
changed.

[0153]A halogen lamp housing 220 provides brightfield illumination for the
sample placed on the sensor 10. The lamp housing 200 directs white light
to a mirror cube 218 and the light is directed through the objective lens
216 of the microscope and onto the sensor 10. Magnified, brightfield
images of the sample are captured by a simple CMOS camera 226. The CMOS
camera 226 allows sample focusing and low-resolution image capture.

[0154]While the variable-angle laser setup of FIG. 12 serves to maximize
the enhanced excitation effect of the device, several other design
elements are provided to optimize the enhanced extraction behavior. In
particular, since the light-emitters on the device (fluorophores, e.g.
quantum dots or organic fluorophores) couple more strongly to the
resonant extraction modes than do autofluorescing materials within the
device itself, attempting to exclude all light except these extraction
modes provides maximum signal-to-noise for the output fluorescence. In
order to accomplish this, there needs to be spatial, frequency, and
polarization filtering. Spatial filtering is accomplished using a low
numerical-aperture (NA) lens 216. While this constrains the extraction
modes to exist within narrow angles of the normal, it provides good
spatial exclusion while still enabling imaging without scanning optics.
(Recall the discussion of FIG. 1 and the strongly directional nature of
the enhanced extraction effect within small angles of vertical). Upstream
magnification (not shown) is employed to overcome the resolution
limitations of imaging with a low-NA lens. Frequency selection is done
with a narrow-linewidth bandpass filter (228) that coincides with the
extraction resonance linewidth. Selecting the polarization for the
resonant light extraction provides further signal-to-noise improvements.

[0155]The fluorescence microscope 195 employs a dichroic mirror in
conjunction with the filter 224 that reflects the excitation laser light
towards a photodiode detector 222 for purposes of measuring the photonic
dispersion of the sensor 10 under test.

[0156]An emission filter 228, as described previously, is used to further
filter the incident fluorescence emission. This filtered output is fed to
a cooled back-thinned electron-multiplier CCD (BT-EM-CCD) 230. This
camera 230 has a very large dynamic range to enable high-enhancement
measurements, and also has excellent sensitivity for pursuing
single-molecule fluorescence detection.

[0157]While presently preferred embodiments have been described with
particularly, variation from the specifics of the disclosed embodiments
are of course possible without departure from the scope of the invention.
All questions concerning scope are to be determined by reference to the
appended claims.